PROJECT SUMMARY ? Project 3 The notoriously immunosuppressive tumor microenvironment (TME) is a major detriment to effective cancer immunotherapy. Pre-clinical evidence in immunocompetent rodent tumor models show that recombinant, non- pathogenic poliovirus (PVSRIPO) can elicit tumor antigen-specific antitumor immunity, in part through reversing immunosuppression and generating an immune-engaged TME. PVSRIPO targets tumor cells and antigen presenting cells (APCs; macrophages/microglia, dendritic cells) via its receptor, the immune checkpoint molecule CD155. PVSRIPO exhibits potent cytotoxic properties in malignant cells, largely due to unhinged PKC-Raf-ERK1/2-MNK signals that provide an enormous advantage to viral protein synthesis. Intriguingly, infection of APCs has a very different outcome. In macrophages or dendritic cells, PVSRIPO infection leads to chronic, non-cytopathogenic viral propagation that produces durable induction of type 1 interferon-dominant stimulation. PVSRIPO-infected APCs exhibit enhanced antigen-presentation and T cell co- stimulation capacity. Viral tumor cell lysis, combined with pro-inflammatory APC stimulation occurring in a context of rampant immune cell invasion into the tumor, set the stage for initiation of antitumor immunity. PVSRIPO has demonstrated promise in an ongoing Phase 1 clinical trial against recurrent WHO stage IV malignant glioma (glioblastoma, gliosarcoma) and was granted Breakthrough Therapy Designation in May, 2016. In the course of this trial, we observed intriguing responses to temozolomide or lomustine in patients that experienced biopsy-proven or imaging-suggested evidence for tumor recurrence post PVSRIPO. We are pursuing the following Aims: 1) Conduct a Phase 2 randomized clinical trial of PVSRIPO alone or in combination with lomustine in patients with recurrent WHO Grade IV malignant glioma. We propose a single-institution, two-arm randomized Phase 2 study that examines the survival of recurrent GBM patients treated with PVSRIPO alone and PVSRIPO + lomustine chemotherapy. Study objectives are: a) to assess the efficacy of a single dose of PVSRIPO with or without a single dose of lomustine among adults with recurrent GBM relative to the survival observed in a (FDA sanctioned) historical control group; b) to assess the safety of PVSRIPO treatment with lomustine; and c) to define changes visualized on imaging due to intratumoral inoculation of PVSRIPO alone or in combination with lomustine. 2) Perform immune monitoring to mechanistically unravel PVSRIPO immunotherapy synergy with lomustine. We will use immune cell profiling and T Cell Receptor (TCR) sequencing to assess: a) global (whole blood) T cell populations; b) effector anti-polio memory T cell populations; c) T cells responding to autologous dendritic cell (DC) stimulus with defined GBM antigens (EGFR, HER2, survivin, hTERT).